POS1017 A NOVEL PAD2/4 BISPECIFIC ANTIBODY BLOCKS PADS ACTIVITY IN VITRO AND DELAYS DISEASE PROGRESS IN CIA-PADS MODEL
Authors: B. Chen, H. Sun, N. Ding, C. Zhan, A. Chen
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Peptidyl arginine deaminase (PAD) catalyzes the conversion of arginine residues to citrulline. With the breakdown of self-tolerance, citrullinated antigens become neo-antigens, potentially triggering autoimmune responses. PAD2 and PAD4 are the most strongly implicated PAD isoforms in RA at both genetic and cellular levels. In RA synovial tissue, neutrophil extracellular traps (NETs) are thought to be the major source of PAD2 and PAD4. However, recent findings indicate that intact neutrophils could secrete PAD2 and express membrane-bound PAD4, which can also catalyze citrullination. Although small-molecule PAD inhibitors such as GSK-484 and AFM-30a have shown good efficacy in PADs enzymatic inhibition assay, no small-molecule PAD inhibitors have entered the clinical stage yet. To avoid off-target effects and disrupting intracellular PAD function, PAD2/PAD4 bispecific neutralizing antibodies may be ideal. Here, we generated a novel and potent PAD2/4 bispecific antibody that is able to block PADs activity both <i>in vitro</i> and <i>in vivo.</i> <h3>Objectives</h3> To develop a novel, highly potent PAD2/4 bispecific blocking antibody to treat rheumatoid arthritis. <h3>Methods</h3> An IgG-ScFv-structure PAD2/4 bispecific antibody was made from hybridoma-derived, humanized PAD2, and PAD4-specific monoclonal antibodies. PAD inhibition was determined by BAEE biochemical assay and ABAP assay using Histone H3 as the substrate. Antibody affinity was measured by Biacore. 15 synovial fluid samples from RA patients were used to test the inhibition efficacy of PAD2, PAD4, and PAD2/4 bispecific blocking antibodies by ABAP assay. The induction of collagen-induced arthritis (CIA) was described previously [1]. CIA-PADs model was induced by immunizing 50 ug human PAD2 and 50 ug human PAD4 together with bovine type II collagen. Each treatment group consisted of 11 mice that were given the PAD2/4 bispecific antibody or control IgG 50 mg/kg i.p. twice a week. <h3>Results</h3> PAD2-mAb, PAD4-mAb, and PAD2/4 bispecific antibodies exhibit high affinity and potent PAD inhibition activity in both BAEE biochemical assay and ABAP assay (Table 1). In RA synovial fluid inhibition assay, the PAD2/4 bispecific antibody showed complete inhibition of PADs activity at 10 nM concentration, but not PAD2-mAb, PAD4-mAb, or GSK484 (Figure 1A), indicating that RA synovial fluid consists of both PAD enzymes. In <i>In vivo</i> models, we demonstrated that co-immunization of PAD2 and PAD4 proteins significantly increased the disease severity, and treatment with the PAD2/4 bispecific antibody decreased the disease activity (Figure 1B). <h3>Conclusion</h3> We have generated a novel anti-PAD2/4 bispecific blocking antibody with potent inhibitory activity in vitro. Furthermore, the blocking activity is also validated in RA patients' synovial fluid and in a novel CIA-PADs model. Additionally, we first demonstrated that co-immunization of PAD2 and PAD4 proteins could increase the disease severity, indicating PAD2 and PAD4 may play an important role in RA disease pathogenesis. In summary, Our PAD2/PAD4 bispecific Ab may provide a novel therapeutic approach towards the treatment of rheumatoid arthritis. <h3>Reference</h3> [1] Willis V C, Gizinski A M, Banda N K, et al. N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis[J]. The Journal of Immunology, 2011, 186(7): 4396-4404. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> BO CHEN Employee of: Qilu Pharmaceutical, He Sun Employee of: Qilu Pharmaceutical, Ning Ding Employee of: Qilu Pharmaceutical, Chuanzong Zhan Employee of: Qilu Pharmaceutical, aqiong chen: None declared.